Hemoglobin-derived blood substitutes are chemically modified hemoglobins that retain, to some degree, the unique oxygen carrying characteristics of red blood cells. In addition to its interaction with oxygen, hemoglobin In both its ferric and ferrous oxidation states interacts with oxides of carbon, nitrogen and sulfur. We are in the process of assembling both equilibrium and kinetic facilities for the study of ligand interaction with hemoglobins. In the meantime, we have carried out a pilot study at DUML (5-9 March, 1990) in which we studied interactions of one such ligand, nitric oxide (NO), with a number of chemically modified hemoglobin using fast reaction techniques. These hemoglobins were crosslinked with the same reagent Diaspirin (bis(3,5-dibromo-salicyl) fumerate) at either the alpha-alpha interface between lys 99 alpha-1, and lys-99 alpha-2 of human deoxy HbA (HbXL99alpha) or at the beta-beta interface between lys-82 beta-2 and lys-82 beta-2 of the oxy human HbA (HBXL 82Beta) and of oxy bovine HbA (Bv HbXL-82 Beta). Rapid mixing experiments of (NO) with ferric forms of these hemoglobins show a biphasic kinetics similar to that of unmodified hemoglobins. However, initial analysis showed an appreciable difference in rates of reaction of modified hemoglobins with (NO). A comprehensive computer generated kinetic analysis of time courses as a function of a number of variables is now under way to determine the effects of various subunit crosslinking on the heme-ligand interactions. (NO) interactions with modified hemoglobins are of particular interest since the endothelial derived relaxing factor (EDRF) Is now believed to be (NO). These experiments provide the framework for in vivo studies on the interactions of EDRF with modified hemoglobins.